Formation of ferrous-CO CBS in this system occurs with concomitant loss of CBS activity as expected. cysteine with homocysteine to give cystathionine and water or H2S respectively (1,2). CBS activity is important for maintaining low steady-state levels of homocysteine, for the biogenesis of cysteine, which limits glutathione synthesis and for production of H2S, a signaling molecule (3C5). Mutations in CBS represent the most common cause of severe hyperhomocysteinemia (6). Crystal structures of CBS (7C9) reveal a considerable (~20 ?) distance between the PLP and heme cofactors, ruling out a direct role for the heme in the reaction mechanism. While a regulatory role for the heme has been suggested, the feasibility of its expression under physiological conditions has been raised, as discussed below (10,11). The heme is six-coordinate in both the ferric and ferrous states and is ligated by His65 and Cys52 in human CBS (7,8,12). A change from the ferric to ferrous heme state is sensed at the PLP site as Amyloid b-Peptide (12-28) (human) evidenced by changes in the chemical shift and line width of the PLP phosphorus resonance (13). A role for heme-based allosteric regulation of CBS is suggested by the observation that perturbation of the heme ligation and/or spin-state is associated with attenuation of enzyme activity (10). Ferrous CBS binds CO with a KD of 1 1.5 0.1 M, which is similar to the affinity for CO of a well-studied heme-based CO sensor, CooA (14C16). However, since the reduction potential for the Fe3+/Fe2+ couple in full-length CBS is low (?350 mV) (11), the Rabbit Polyclonal to PTTG physiological relevance and reversibility of CO-based inhibition have remained open questions. In this study, we demonstrate for the first time, coupled reduction-carbonylation of CBS in the presence of CO and a physiologically relevant reducing partner, i.e. human methionine synthase reductase (MSR), an NADPH-dependent cytosolic diflavin oxidoreductase. Formation of ferrous-CO CBS in this system occurs with concomitant loss of CBS activity as expected. Importantly, CO removal or air oxidation of ferrous-CO CBS leads to recovery of the active ferric form and demonstrates the reversibility of the heme-dependent regulatory switch being modulated by a physiological reducing system. MSR serves as a conduit for electrons from NADPH through FAD and FMN to methionine synthase and to surrogate electron acceptors (17). In the presence of CO, NADPH and substoichiometric MSR, conversion of ferric CBS with a Soret maximum at 428 nm and broad / bands (centered at ~550 nm), to the ferrous CO species with a Soret maximum at 422 nm and sharpening of the / bands at 570 and 540 nm respectively, is observed (Fig. 1). Formation of ferrous-CO CBS is not observed if any of the assay components is omitted. The isosbestic conversion of ferric to ferrous-CO CBS indicates that the ferrous intermediate does not accumulate to detectable levels. This is consistent with the ~120 mV potential difference that separates the FMN semi-quinone/hydroquinone (?227 mV) (18) and the CBS ferric/ferrous redox couples (11). We postulate that kinetic coupling between reduction and carbonylation of the heme traps the ferrous intermediate and shifts the unfavorable equilibrium for the reduction to the right (Scheme 1). Open in a separate window Fig. 1 Spectral changes associated with MSR-dependent reductive carbonylation and air oxidation of CBS. Human CBS (5 M) in 100 mM anaerobic CO-saturated potassium phosphate buffer, pH 7.4, was mixed with 0.5 M human MSR and 500 M NADPH to generate the ferrous CO form. The latter converted to ferric CBS upon exposure to air. Open in a separate window Scheme I CBS heme oxidation and ligation states (A) and PLP tautomeric states (B) CO displaces Cys52 as Amyloid b-Peptide (12-28) (human) the heme ligand in human CBS (19,20) and this is accompanied by inhibition of enzyme activity with a Ki of 5.6 M (21). Formation of ferrous-CO CBS in the Amyloid b-Peptide (12-28) (human) presence of reduced MSR also inhibits CBS activity in the standard assay (92 6 mole cystathionine formed mg?1 h?1). The reversibility of CO inhibition was tested by air-oxidation of the ferrous-CO CBS sample. The Soret absorption maxi mum at 428 nm indicated recovery of the ferric CBS form (Fig. 1). The activity of the oxidized enzyme was 344 6 mole mg?1 h?1, which is comparable to that of the starting ferric CBS sample.